Neutrophil responses are a critical element in host defense during, for example, bacterial infections, but neutrophil responses can also be overtly pathogenic. Thus, in many inflammatory diseases neutrophils contribute more to the pathology than do the microbes themselves (Nathan, 2002, Nature 420:846-852). Neutrophil recruitment is a complex process involving activation of local structural cells (e.g., epithelial cells) via pathogen-associated molecular pattern receptors, complement cascade products or arachidonic acid metabolites, such that the structural cells express inflammatory mediators (e.g., IL-1, CXCL8) (Smith et al., 2001, J. Immunol. 167:366-374). These can in turn activate regional endothelial cells, which actively foster extravasation of neutrophils via the now classical paradigm of chemokine- (e.g., CXCL8) and adhesion molecule-mediated rolling-arrest-diapedesis (Baggiolini, 1998, Nature 392:565-568; Springer, 1994, Cell 76:301-314).
The ELR-CXC chemokines are a subgroup of the CXC chemokine family in which the amino sub-terminal two cysteine residues (i.e., CXC), which are separated by an alternate amino acid, are immediately preceded by a Glu-Leu-Arg (i.e., ELR) motif. They include CXCL1-3 and 5-8 (growth-related oncogene α, β, and γ [GRO α, β, and γ], epithelial cell neutrophil-activating peptide-78 [ENA-78], granulocyte chemotactic protein-2 [GCP-2], neutrophil activating peptide-2 [NAP-2], and interleukin-8 [IL-8], respectively)(Baggiolini, 1998, Nature 392:565) which chemoattract and activate neutrophils via two closely related G protein-coupled receptors (GPCR), the CXCR1 and CXCR2. CXCL8 binds both receptors with high affinity, while CXCL6 binds both receptors with lower affinity (Wolf et al., 1998, Eur. J. Immunol. 28:164-170; Wuyts et al., 1998, Eur. J. Biochem. 255:67-73). The other ELR-CXC chemokines bind to the CXCR2, also with relatively lower affinities (Wuyts et al., 1998; Ahuja and Murphy, 1996, J. Biol. Chem. 271:20545-20550). Both the CXCR1 and CXCR2 can trigger chemotactic responses and intracellular Ca2+ flux in neutrophils and contribute to elastase release (Wuyts et al., 1998; Chuntharapai and Kim, 1995, J. Immunol. 155:2587-2594), but activation of the respiratory burst and phospholipase D release responses are reportedly CXCR1-dependent (Jones et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:6682-6686), whereas CXCR2 signaling is critical to MMP-9 release (Chakrabarti and Patel, 2005, J. Leukoc. Biol. 78:279-288). Moreover, these two receptors may be differentially involved in neutrophilic pathology in vivo. For example, the CXCR1 is reportedly of more importance in inflammatory bowel diseases (Gijsbers et al., 2004, Eur. J. Immunol. 34:1992-2000), sepsis and acute respiratory distress syndrome (Cummings et al., 1999, J. Immunol. 162:2341-2346; Goodman et al., 1999, Chest 116:111s-112s), while it and the CXCR2 both play roles in synovial infiltration by neutrophils in arthritic joints (Podolin et al., 2002, J. Immunol. 169:6435-6444). However, numerous alternate GPCRs are involved in neutrophilic inflammation, including those for LTB4, C5a, and fMLP, such that antagonism of either LTB4 or C5a has been shown to be of significant benefit in various inflammatory settings (Park et al., 2000, Anesth. Analg. 89:42-48; Crooks at al., 2000, Eur. Respir. J. 15:274-280; Wollert et al., 1993, Surgery 114:191-198). The precise interrelationships between these mediators in inflammatory responses have not been formally determined, but it has been reported that signaling through the C5a or fMLP receptors can effectively desensitize the ELR-CXC chemokine receptors (Blackwood et al., 1996, J. Leukoc. Biol. 60:88-93). On the other hand, CXCL8 reportedly poorly desensitizes some events (e.g., intracellular Ca++ flux) associated with C5a and fMLP receptor signaling (Richardson et al., 1995, J. Biol. Chem. 270:27829-27833; Tomhave et al., 1994, J. Immunol. 153:3267-3275), although it can desensitize chemotactic responses driven by these ligands.
There are an array of inflammatory settings in which neutrophils are the primary drivers of host pathology and for some of these one or more ELR-CXC chemokines have been implicated in the neutrophil response. When activated, neutrophils release an array of microbicidal factors, including reactive oxygen intermediates (ROI), defensins, and proteolytic enzymes, but they also foster the inflammatory responses through elaboration of proinflammatory cytokines (e.g. TNF, IL-1) and ELR-CXC chemokines themselves (e.g., CXCL1, CXCL8). Aspiration pneumonia, brought on by aspiration of highly acidified gastric contents into the lungs, occurs primarily in unconscious or semiconscious patients. Its incidence is approximately 1 in 3000 during general anaesthesia surgeries (Olsson et al., 1986, Acta Anaesthesiol Scand. 30:84-92; Warner et al., 1993, Anesthesiology 78:56-62). The local inflammatory sequel varies from sub-clinical pneumonitis to severe acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (Pepe at al., 1982, Am J Surg 144:124-30; Fowler et al., 1983, Ann Intern Med 98:593-7) depending on the volume and the pH of the gastric contents (Marik, 2001, N Engl J Med 344:665-71). However, ALI/ARDS associated with aspiration pneumonia carries a mortality rate of 10-30% (Olsson et al., 1986; Warner et al., 1993). It is recognized that neutrophils are the primary drivers of the inflammatory cascade in aspiration pneumonia (Beck-Schimmer et al., 2005, Anesthesiology 103:556-66; Raghavendran et al., 2005, Am J Physiol Lung Cell Mol Physiol 289:L134-43; Folkesson et al., 1995, J Clin Invest 96:107-16; Davidson et al., 2005, Am J Physiol Lung Cell Mol Physiol 288:L699-708) and that the ELR-CXC chemokines are central to the recruitment and activation of neutrophils in aspiration pneumonia (Beck-Schimmer et al., 2005; Folkesson et al., 1995; Rotta et al., 2004, Crit. Care Med 32:747-54). This suggests that, in the case of aspiration pneumonia, for example, ELR-CXC chemokine antagonism could be an ideal therapeutic approach to ameliorate pathology, and this applies also to many neutrophilic inflammatory disorders (Walley et al, 1997, Infect Immun 65:3847; Kishimoto et al, 2001, J Virol 75:1294; Mukaida et al, 1998, Inflamm Res 47 (Suppl 3):S151; Jones et al, 1997, J Biol Chem 272:16166; White et al, 1998, J Biol Chem 273:10095)
Previously, we generated a broad-spectrum ELR-CXC chemokine antagonist bovine CXCL8(3-74)K11R/G31P (bG31P) (Li and Gordon, 2001, Biochem Biophys Res Commun 286:595-600; Li et al., 2002, Biochem Biophys Res Commun 293:939-44), which blocks the ability of ELR-CXC chemokines to activate and chemoattract neutrophils in vitro (Li et al., 2002). A single treatment with bG31P≈97% blocks neutrophil infiltration into intradermal endotoxin challenge sites for 2-3 days (Li et al., 2002, Vet Immunol Immunopathol 90:65-77), and dramatically reduces pulmonary pathology and pyrexia in animals suffering from airway endotoxin challenge (Gordon et al., 2005, J Leukoc Biol 78:1265-72). In the process of generating a human homologue of bG31P we developed multiple human-bovine chaemeric forms of bG31P that were also effective in antagonizing ELR-CXC chemokine-mediated airway endotoxin-induced pathology (Zhao et al., 2007, Internat Immunopharmacol 7:1723-31), and more recently we engineered a fully human form of G31P, human CXCL8(3-72)K11R/G31P (G31P). This latter drug not only blocks neutrophil activation induced by ELR-CXC chemokines, but also human airway epithelial cell responses driven by bacterial lipopolysaccharide (LPS). Importantly, it also partially antagonizes heterologous GPCRs that are involved in neutrophilic inflammation, including those for C5a, LTB4, and fMLP (Zhao et al, 2009, J Immunol 182:3213). We subsequently showed that G31P can highly effectively reduce lung pathology in a guinea pig model of aspiration pneumonia, and that it did so without negatively impacting bacterial clearance in the lungs of the affected animals (Zhao et al, 2010, Pulm Pharmacol & Therap 23:22)(see also FIGS. 1-5).
Neutrophilic inflammation is also thought to be critical to both local and remote organ injury following mesenteric artery ischemia-reperfusion (I/R) injury. Intestinal I/R injury is associated with severe multiple organ failure (MOF), including the lung as one such remote organ (He et al., 2008, PLoS ONE 3: e1527; Schmeling et al., 1989, Surgery 106: 195-201; Caty et al., 1990, Ann Surg 212: 694-700). A number of proinflammatory cytokines and chemokines are critically involved in this pathology (Caty at al., 1990) although the pathogenesis of intestinal I/R-induced gut pathology and MOF is complex and not completely understood. Nonetheless, reactive oxygen intermediate (ROI) generation and neutrophil sequestration are believed to be two fundamental causative factors (Schmeling et al., 1989; Cerqueira et al., 2005, Acta Cir Bras 20: 336-343; Souza et al., 2004 Br J Pharmacol 143: 132-142). The pivotal role of neutrophils in these processes (Schmeling at al., 1989; Cerqueira et al., 2005; Bless et al., 1999, Am J Physiol 276: L57-63) suggests that, here too, blockade of their recruitment could be an important therapeutic approach to intestinal I/R injury. The list of neutrophil agonists potentially involved in I/R injury includes TNFα and IL-β (Caty et al., 1990) and the ELR-CXC chemokines (e.g., CXCL1, CXCL8, MIP-2) (Schmeling, et al., 1989; Bless et al., 1999; Sekido et al., 1993, Nature 365: 654-657), but also C5a (Bless at al., 1999; Wada et al., 2001, Gastroenterology 120: 126-133), LTB4 (Souza at al., 2000, Eur J Pharmacol 403: 121-128), ROI (Koike et al., 1993, J Surg Res 54: 469-473), and adhesion molecules (Bless at al., 1999). Multiple reports have implicated individual ELR-CXC chemokines as primary effectors and shown that their neutralization ameliorates I/R-induced local and remote organ pathology (Souza et al., 2004; Bless et al., 1999; Miura et al., 2001, Am J Pathol 159: 2137-2145; Kaneko et al., 2007, Eur Surg Res 39: 153-159), making these mediators attractive therapeutic targets. Given this broad panel of inflammatory mediators (many of which employ GPCR as their cellular receptors) implicated in intestinal I/R injury, the above-noted abilities of G31P to induce desensitization of heterologous GPCR raises the question of whether G31P would be particularly useful as a therapeutic approach in this disease. Thus, we assessed this in a rat model of superior mesenteric artery (SMA) I/R injury, treating the animals with G31P and examining their local and remote organ pathology in this model. We found that G31P reduced mortality in the I/R rats by 50% relative to saline-treated I/R animals, and that it reduced local and remote organ injury (Zhao et al, 2010, J Surg Res 162:264)(see also FIGS. 6-12).
Some of the protective effects of bG31P in airway endotoxemia, for example, seemed to be somewhat tangentially related to its putative neutrophil-centered effects. For example, bG31P treatment reduces endogenous pyrogen expression and pyrexia prior to the time when neutrophils were appreciably present in the airways, suggesting that it may have effects on structural (e.g., epithelial) cells. Moreover, data from a study of its effects in aspiration pneumonia, wherein high level bacterial colonization of the lungs occurs, confirmed its effectiveness in this environment as well. In such cases the bacteria would release formyl peptides (e.g., fMLP) among other products and activate the complement cascade, leading to the generation of C5a. Thus, formally explored further the mechanisms by which G31P might interact with airway epithelial cells, but also its effect on neutrophil responses to ligands for heterologous GPCR, and its specificity for the CXCR1 and CXCR2 (Zhao et al, 2009, J Immunol 182:3213) (See also FIGS. 12-19).
Just as the ELR-CXC chemokines can activate neutrophils and epithelial cells, there is abundant evidence that they have important roles in the biology of other types of cells, including transformed (i.e., cancer) cells. ELR-CXC chemokines such as CXCL1, CXCL6, or CXCL8 are secreted by a variety of human tumor cells and are involved in a number of important tumor-related biological processes, including tumor formation, development, and responses to chemotherapy in the context of melanoma, ovarian, prostate, pancreatic, bladder, lung and breast cancers (Araki et al., 2007, Cancer Res 67:14; Luppi et al., 2007, Lung cancer 56: 25-33; MacManus et al., 2007, Mol Cancer Res 5; Zhu et al, 2006, Br J Cancer 94:1936; Oladipo et al, 2011, Br J Cancer 104:480). Indeed, the levels at which CXCL8, for example, are expressed by prostate cancer PC-3 cells, for example, correlates well with the extent to which the cells develop into tumors, promote neovascularization, and metastasize following orthotopic implantation into nude mice (Kim et al, 2001, Neoplasia 3:33). Significantly, CXCR1 and CXCR2 are expressed at high levels in many tumour cells including prostate cancer cells (Kim et al, 2001), melanoma (Singh et al, 2010, Future Oncol 6:111; Singh et al, 2009, Br. J. Cancer 100:1638), and adenocarcinoma (Varney et al, 2011, Cancer Left 300:180) cells, and in each case these receptors contribute importantly to tumour growth, metastasis and angiogenesis (Kim et al, 2001; Singh et al, 2010; Singh et al, 2009; Varney et al, 2011).
Herein, we tested whether ELR-CXC chemokine antagonism with G31P would by itself be beneficial in mouse models of melanoma, hepatic adenocarcinoma, and orthotopic human prostate cancer. We assessed the impact of G31P on a number of tumor cell parameters, including chemokine-driven tumour cell proliferation, in vitro. We also assessed the impact of G31P on tumor growth, metastasis and angiogenesis in vivo